HLA-B*2736等位基因的序列分析

使用FLOW-SSO、PCR-SSP以及测序等分型技术, 发现一个与HLA-B*270401基因相关的未知基因。设计基因特异性引物单独扩增B*27基因的外显子2-5, 包括内含子2-4, 并进行双向测序, 分析与B*270401基因序列的差异。该基因的扩增产物为1 815 bp。与B*270401相比在外显子3和4共有10个碱基的改变, 从而使相应氨基酸发生错义或同义突变。碱基634 A→C (密码子130丝氨酸→精氨酸); 670 A→T (密码子142苏氨酸→丝氨酸); 683 G→T (密码子146色氨酸→亮氨酸); 698 A→T (密码子151谷氨酸→缬氨酸); 774 G→C (密码子176谷氨酸→天冬氨酸); 776 C→A (密码子177苏氨酸→赖氨酸); 781 C→G (密码子179谷氨酰胺→谷氨酸); 789 G→T (密码子181丙氨酸同义突变); 1 438 C→T (密码子206甘氨酸同义突变); 1 449 G→C (密码子210甘氨酸→丙氨酸)。在IMGT/HLA数据库中B*27组只有3个基因（B*270502 / 2706 / 2732）提交了内含子序列。该未知基因的内含子2序列与B*2706相同, 显示了与B*27组基因的同源性, 但其同源性在内含子3、4均未得到支持, 与B*27组基因相比, 内含子3的第106个碱基C→G, 碱基168缺失, 碱基179 G→A, 碱基536 G→A; 内含子4中碱基82 T→C。但其内含子3、4序列却与B*070201完全相同。该基因序列已提交GenBank, 编号为被DQ915176, 被WHO确认为HLA-B*2736等位基因。     

Aberrant expression of HLA-B*3565Q is associated with a disrupted disulfide bond

The identification of expression variants is a challenge in HLA diagnostics. We here describe the identification of the novel allele HLA-B*3565Q. The serological HLA class I type, as determined by a lymphocytotoxicity test, was A11,24; B38; Bw4; Cw−; whereas PCR-sequence-specific primers resulted in A*11,*24, B*35,*38; Cw*12, thus suggesting the presence of a nonexpressed B*35 allele. To clarify the lack of serological HLA-B35 reactivity, exons 2 and 3 were sequenced following haplotype-specific amplification. At position 564 from the beginning of the coding region (exon 3), a transversion (C→G) was observed, which, at the amino acid level, results in a substitution from cysteine to tryptophane at position 164 of the mature polypeptide. Because this position is essential for the formation of a disulfide bond linking the cysteine residues at positions 101 and 164, which is strongly conserved in functional class I molecules of vertebrates, the disruption of this bond is very likely to be the reason for the lack of serological detectability. We later found the same novel allele in a second unrelated individual, of whom we were able to establish a lymphoblastoid cell line (B-LCL). Serological testing of this B-LCL indicated a very low aberrant expression of HLA-B*3565Q, which cannot be expected to be detected by standard serology techniques. Holger-Andreas Elsner and Peter A. Horn contributed equally to this work. The nucleotide sequence data reported in this paper have been published in the European Molecular Biology Laboratory, GenBank, and DNA Data Bank of Japan Nucleotide Sequence Database under the accession numbers AJ278746, AJ278747, and AJ879892. The name B*3565Q was officially assigned by the WHO Nomenclature Committee in December 2005. This follows the agreed policy that, subject to the conditions stated in the most recent Nomenclature Report (Marsh et al. 2005), names will be assigned to new sequences as they are identified. Lists of such new names will be published in the following WHO Nomenclature Report.     

Comparison of abacavir-specific effector and proliferating functions of CD8 T cells in abacavir-treated HIV-1 patients

Susceptibility to abacavir hypersensitivity (ABH) in HIV-1-positive patients is strongly linked to the carriage of HLA-B*57:01 and the potential mechanism includes drug-specific activation of cytokine producing CD8 T cells exclusively in individuals carrying HLA-B*57:01. Here, we report a detailed characterization of abacavir-induced functional response of CD8 T cells in HLA-B*57:01^pos individuals. Peripheral blood mononuclear cells (PBMNCs) from HLA-B*57:01^posABH^pos and HLA-B*57:01^negABH^neg individuals were stimulated with abacavir. Multicolor flow cytometry was performed to assess the cytokine (IFNγ) production and degranulation (CD107a expression) after 6–18 hr culture and to enumerate proliferating CD4/CD8 T cells by culturing carboxyfluorescein diacetate succinimidyl ester-loaded PBMNCs for 7 days. CD8 T cells from HLA-B*57:01^posABH^pos individuals were multifunctional: proliferating, IFNγ producing, degranulating (CD107a^pos), and both degranulating and IFNγ producing (CD107a^posIFNγ^pos). Degranulating CD8 T cells in general and both degranulating and IFNγ producing CD8 T cells in particular dominated abacavir-specific immune response. All functional responses were partially blocked by addition of HLA-B*57:01-reactive Bw4 mAb, but not by non-HLA-B*57:01-reactive Bw6 mAb. In conclusion, the study demonstrates that abacavir-specific CD8 T-cell-restricted immune response in HLA-B*57:01^posABH^pos HIV-1 patients has multiple effector and proliferating functions, where the primary effector response appears to be the release of cytolytic granules. The findings have implications for immunotherapy of HLA-related drug hypersensitivities.     

MIC-A allele profiles and HLA class I associations in Behçet's disease

Recently a new family of non-classical MHC molecules, the MHC class I chain-related protein (MIC), encoded by genes located in the major histocompatability complex have been identified. On the basis of the location of MIC genes and the structure and expression of MIC molecules it has been postulated that MIC may be a susceptibility factor in Behçet's disease (BD). We investigated the association of the 16 described external domain alleles and the transmembrane triplet repeats of MIC-A with BD in a Middle Eastern population. DNA from ninety-five patients and 102 age- and sex-matched controls were analyzed by polymerase chain reaction using allele specific primers. Our results show an increase of MIC-A*009 in the BD patient group 44/95 (46%) compared with controls 24/102 (24%) (χ²=11.3, OR=2.8, P=0.00078). MIC-A*009 was also found to be strongly associated with HLA-B51 in the patients 39/44 (88%) when compared with controls 10/24 (42%) (χ²=4, P=0.04). MIC-A*009 was also found in linkage disequilibrium with HLA-B52, but only in controls. The A6 form of a MIC-A transmembrane triplet repeat was found to be significantly raised in the patients (80/95; 84%;) compared with controls (58/102, 57%) (χ²=17.5, OR=4, P=0.000028). Although the MIC-A associations described are highly significant, the association with HLA-B51 independently remains the most significant factor (χ²=56.8, P<10^–6). The data suggests that as both MIC-A*009 and A6 are in strong linkage disequilibrium with HLA-B51, they are unlikely to be the susceptibility gene for BD but may be markers for additional risk factors.     

The Pharmacogenomic HLA Biomarker Associated to Adverse Abacavir Reactions: Comparative Analysis of Different Genotyping Methods

Many pharmacogenomic biomarkers (PGBM) were identified and translated into clinical practice, affecting the usage of drugs via label updates. In this context, abacavir is one of the most brilliant examples of pharmacogenetic studies translated into clinical practice. Pharmacogenetic studies have revealed that abacavir HSRs are highly associated with the major histocompatibility complex class I. Large studies established the effectiveness of prospective HLA-B*57:01 screening to prevent HSRs to abacavir. Accordingly to these results the abacavir label has been modified: the European Medicines Agency (EMA) and the FDA recommend/suggested that the administration of abacavir must be preceded by a specific genotyping test. The HLA locus is extremely polymorphic, exhibiting many closely related alleles, making it difficult to discriminate HLA-B*57:01 from other related alleles, and a number of different molecular techniques have been developed recently to detect the presence of HLA-B*57:01. In this review, we provide a summary of the available techniques used by laboratories to genotype HLA-B*57:01, outlining the scientific and pharmacoeconomics pros and cons.     

双通道TaqMan实时荧光PCR应用于别嘌呤醇不良反应相关基因HLA-B*58:01的检测与验证

摘要 目的：建立一种双通道TaqMan实时荧光PCR方法,应用于别嘌呤醇不良反应相关基因HLA-B*58:01的快速基因检测,并验证该方法的灵敏度、特异性、重复性及可靠性等性能指标。方法：针对HLA-B*58:01 等位基因序列设计引物和Taqman 探针,建立一种双通道TaqMan实时荧光PCR检测方法,收集陕西省人民医院、浙江大学医学院附属第二医院、浙江大学医学院附属邵逸夫医院三家医院收治的1158例临床诊断为痛风或血清尿酸高患者的外周血标本,采用双盲对照实验,分别采用双通道TaqMan实时荧光PCR法与已获审批的商业试剂盒共同检测所有标本,对比两种方法的检测结果进而评价该TaqMan实时荧光PCR方法的临床性能。结果：双通道TaqMan实时荧光PCR法特异性高,稳定可靠,可检测低至1 ng/?滋L的阳性样本;采用双通道TaqMan实时荧光PCR法与已获审批的商业试剂盒在1158份患者标本中检出的HLA-B*58:01阳性及阴性标本均相同,阳性标本147例,阴性标本1011例,该组患者HLA-B*58:01基因携带率为12.69%,两种方法符合率为100%（1158/1158）,两组实验结果数据采用SPSS分析P=1.000,差异无统计学意义。所有受检者HLA-B* 58:01携带率为12.69 % （147 /1158）。结论：双通道TaqMan实时荧光PCR可以作为一种快速、特异、灵敏的HLA-B*58:01的基因检测方法,用于别嘌呤醇用药前的相关不良反应风险评估。     

HLA-B27与强直性脊柱炎免疫学机制的研究进展

强直性脊柱炎是与HLA-B27有明确相关性的自身免疫性疾病,其发病过程包括了微生物和宿主的相互作用、免疫细胞对MHC-I类分子的识别以及细胞因子网络的失平衡等方面.未折叠蛋白应答反应参与了强直性脊柱炎发病过程,并且下游IL-23/IL-17轴的激活可能在发病过程中起重要作用.未折叠蛋白反应和IL-23/IL-17轴是研究强直性脊柱炎发病机制和防治措施的新靶点.     

Dissection and molecular analysis of alloreactive CD8+ T cell responses in allogeneic haematopoietic stem cell transplantation

We applied a cDNA expression screening procedure with cryopreserved non-clonal CD8+ T cell populations (Lennerz et al., Proc. Natl. Acad. Sci. USA 102:16013-8, 2005) to the identification of candidate antigens for graft-versus-host disease (GvHD) and graft-versus-leukaemia (GvL) effects in allogeneic haematopoietic stem cell transplantation (allo-HSCT). In a patient–donor model system with HLA class I disparities, we identified an HLA-B*44 mismatch allele, HLA-B*4405, as the dominant target of alloreactive T cells expanded in vitro from donor peripheral blood mononuclear cells (PBMC). HLA-B*4405-reactive T cells were detectable after multiple in vitro stimulations in the patient’s post-HSCT PBMC. In a patient–donor model with full HLA compatibility, the major target antigen of donor lymphocytes stimulated in vitro with the respective patient’s pre-HSCT PBMC was restricted by HLA-A*0201 and was encoded by TRIM22-442 C, a newly detected polymorphic allele of the tripartite motif family member TRIM22 (synonym: STAF50), preferentially expressed in cells of the haematopoietic system. An arginine(R)-to-cysteine(C) exchange at position 442 generated an immunogenic T cell epitope equivalent to a minor histocompatibility antigen (mHag). TRIM22-442C-specific T cells persisted long-term in the patient’s post-HSCT PBMC. Approximately, 1.3% of Caucasians carry TRIM22.442 C in association with HLA-A*0201. In particular, the knowledge of a large and diverse panel of such mHags may be crucial for further improvement of donor selection and adoptive T cell transfer strategies. The procedure applied herein will help to accelerate and facilitate their identification.     

Substantial Influence of ERAP2 on the HLA-B*40:02 Peptidome: Implications for HLA-B*27-Negative Ankylosing Spondylitis

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Highlights

• •HLA-B*40:02 and ERAP2 are risk factors for ankylosing spondylitis.
• •The effects of ERAP2 on the B*40:02 peptidome are defined.
• •ERAP2 has a major influence mainly due to alterations of N-terminal residues.
• •These effects provide a basis for the association of ERAP2 with disease.

     

hβ2m/HLA-B2704双转基因小鼠的制备及β2m对HLA-B2704表达的影响

制备hβ2m HLA B2 70 4双转基因小鼠 ,研究hβ2m对HLA B2 70 4表达的影响 .通过hβ2m转基因小鼠和HLA B2 70 4转基因小鼠交配 ,出生动物及后代经PCR初步筛选 ,采用Southern杂交对经PCR初步筛选的hβ2m HLA B2 70 4双转基因阳性小鼠基因组DNA标本作进一步鉴定 .阳性者进行RT PCR和流式细胞光度术检测其在mRNA和蛋白水平的表达 .hβ2m阳性小鼠和HLA B2 70 4阳性小鼠交配 ,生仔鼠 6 7只 ,其中 2 8只仔鼠同时整合hβ2m和HLA B2 70 4基因 .Southern杂交证实 ,阳性鼠同时都含有hβ2m和HLA B2 70 4基因 .阳性小鼠的皮肤、结肠、睾丸和脾脏组织中均有HLA B2 70 4和hβ2m基因的mRNA表达 ,其外周血淋巴细胞膜上HLA B2 70 4基因的蛋白表达为 35 87% ,在HLA B2 70 4单转基因小鼠外周血淋巴细胞膜上HLA B2 70 4基因的蛋白表达为 7 87% (Histogramsta tistics) .成功地制备了hβ2m HLA B2 70 4双转基因小鼠 .在hβ2m HLA B2 70 4双转基因小鼠的外周血淋巴细胞膜上 ,HLA B2 70 4基因在蛋白水平表达较高 ,而在HLA B2 70 4单转基因小鼠中则表达不明显 .hβ2m可与HLA B2 70 4重链分子结合 ,稳定和增高其在淋巴细胞膜上的表达     

用组合肽库技术分析HLA-B27的抗原提呈模式

HLA分子抗原表位提呈模式的分析,在自身免疫病和肿瘤的病因与治疗研究方面有重要意义。本研究采用组合肽库的策略合成19组ORX7型肽亚库,通过与荧光素标记肽的竞争结合试验,分析了与强直性脊柱炎有强相关的HLA-B27分子的抗原提呈模式。结果显示HLA-B27与P1为不同氨基酸残基的19种肽亚库有相近的结合率,提示P1为非锚定残基;中国人群最常见的二种HLA-B27亚型B*2704和B*2705,在提呈肽表位的P1模式方面存在一些小差异,P1为D或E的肽亚库与HLA-B*2704的结合能力要强一些,而P1为K的肽亚库则与HLA-B*2705的结合能力强一些。本研究为HLA-B27与强直性脊柱炎关联机制的研究提供了线索,为开展HLA分子的抗原提呈模式分析打下了基础。     

Structural modeling of HLA-B*1502/peptide/carbamazepine/T-cell receptor complex architecture: implication for the molecular mechanism of carbamazepine-induced Stevens-Johnson syndrome/toxic epidermal necrolysis

Drug-induced adverse reactions are a significant problem in healthcare worldwide and are estimated to cost billions of dollars annually in the United States. A portion of such reactions is observed to strongly associate with certain human leukocyte antigen (HLA) alleles; one of the strongest associations is the HLA-B*1502 protein with carbamazepine (CBZ)-induced Stevens–Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) – the odds ratio value can even be higher than one thousand. The particularly strong association in CBZ-induced SJS/TEN suggests that the HLA-B*1502 is not only a genetic marker but also a participant in the pathogenesis of the disease. In the current study, we attempt to computationally model the atomic-level structure of the complete HLA-B*1502/peptide/CBZ/T-cell receptor (TCR) complex architecture based on prior knowledge obtained from epidemiological investigations as well as in vitro and in vivo assays. The model tells a different story about the molecular mechanism of CBZ-induced SJS/TEN from that previously reported for abacavir (ABC)-induced hypersensitivity (HSR); the CBZ molecule is located at the interface between HLA-B*1502/peptide and TCR, directly contacts the P3–P6 residues of antigen peptide, and bound within a pocket region encompassed by two TCR CDR3 fingers. Molecular dynamics simulation and binding energy analysis further reveal that the CBZ shows considerably high affinity to TCR over HLA-B*1502/peptide, which can tightly interact with the former rather than the latter. From the model, two hypotheses are proposed that can well explain most previous observations and are expected to guide next wet-lab experiments. This study could help to promote our understanding of the molecular mechanism and pathological implication underlying CBZ-induced SJS/TEN.     

Residue 116 determines the C-terminal anchor residue of HLA-B*3501 and -B*5101 binding peptides but does not explain the general affinity difference

 HLA-B^*3501 and -B^*5101 molecules, which belong to the HLA-B5 cross-reactive group, bind peptides carrying similar anchor residues at P2 and the C-terminus, but differences are observed in the preference for a Tyr residue at the C-terminus and the affinity of peptides. A recent study of HLA-B^*3501 crystal structure suggested that residue 116 on the floor of the F-pocket determines a preference for anchor residues at the C-terminus. In order to evaluate the role of the residue 116 in the peptide binding to both HLA-B^*3501 and HLA-B^*5101 molecules, we generated HLA-B^*3501 mutant molecules carrying Tyr at residue 116 (B^*3501–116Y) and tested the binding of a panel of nonamer peptides to the B^*3501–116Y molecules by a stabilization assay with RMA-S transfectants expressing the mutant molecules. The substitution of Tyr for Ser at residue 116 markedly reduced the affinity of nonamer peptides carrying Tyr at P9, while it enhanced that of nonamer peptides carrying Ile and Leu at P9. On the other hand, the affinity of peptides carrying aliphatic hydrophobic residues at P9 to B^*3501–116Y molecules was much higher than that to HLA-B^*3501 and HLA-B^*5101 molecules. These results indicate that residue 116 is critical for the structural difference of the F-pocket between HLA-B^*3501 and HLA-B^*5101 which determines the C-terminal anchor residues, while leaving other residues which differ between HLA-B^*3501 and HLA-B^*5101 may be responsible for the low peptide binding property of the latter. Received: 18 April 1997 / Revised: 18 September 1997     

Molecular genetic analysis of weak B blood group allele in Jinan population reveals distribution of ABw phenotype

The aim of this study was to investigate the distribution of the ABw phenotype of ABO blood group in the Jinan population. 31 856 samples were tested during the period 2018 to 2019. Thirty-nine samples with discrepant results, as identified by micro-column gel method, were further investigated by serological (tube technique) and molecular (fluorescence PCR, DNA sequencing) methods. Eight samples showed ABw phenotype, which accounted for 0.025% of the population tested. From the sequencing analysis, six samples (6/8) were typed as ABO*A1.02/ABO*BW.12 and two samples (2/8) as ABO*A1.02/ABO*BW.03. The study suggests that ABw12 account for 75% of ABw phenotype and indicate ABw12 is the main ABw phenotype in Jinan population.     

HLA class II alleles with susceptibility of leprosy in the Mexican Mestizo population

Leprosy is a chronic, infectious disease, caused by Mycobacterium leprae, Mycobacterium lepromatosis or both, which affects the peripheral nervous system and the skin. Activation of cellular immunity in infected individuals depends on antigen recognition, which involves relevant HLA-Class II alleles. Therefore, the objective of this study was to determine HLA-Class II allele frequencies (HLA-DRB1and-DQB1)in Mexican Mestizo leprosy patients and compare themwith healthy controls, in order to define their role in the genetic susceptibility to this infection.The genomic DNA of each participant was obtained from peripheral blood, using the salting-out method. PCR amplification and hybridization ofHLA-class II alleleswas made by PCR-SSO. The results showed that frequencies of HLA-DRB1*15(Pc =0.003, OR=3.3 95%CI=1.53-7.33), HLA-DQB1*05(Pc =0.00003, OR=6.03 95%CI=2.49-14.61) and HLA-DQB1*06 (Pc =0.007, OR=2.89, 95%CI=1.38-6.04)were significantly higher among leprosypatients than those of healthy controls. The study suggests that HLA-DRB1*15, HLA-DQB1*05, and HLA-DQB1*06 are associated with leprosy susceptibility in the Mexican Mestizo population.     

Redundancy and Complementarity between ERAP1 and ERAP2 Revealed by their Effects on the Behcet's Disease-associated HLA-B*51 Peptidome,

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Highlights

• •HLA-B*51 and ERAP1, but not ERAP2, are risk factors for Behçet's disease.
• •The HLA-B*51 peptidome and the effects of ERAP1 and ERAP2 on it are analyzed.
• •ERAP1 and ERAP2 alter multiple features of the HLA-B*51 peptidome in distinct ways.
• •Both enzymes act independently with complementary and partially redundant functions.

     

物种对资源竞争的动力机制及数值模拟

建立了集合种群物种在两个斑块中对资源竞争的数学模型,并进行了数值模拟实验,结果表明：（1）通过R^＊来预测竞争物种的结局,存在几种可能性：一是具有低R^＊值的物种竞争取代高R^＊值的物种;二是具有不同R^＊值的物种,甚至是具有相同R^＊值的物种也存在共存的可能性;三是具有高R^＊值的物种也可以竞争排斥低R^＊值的物种,结论存在不确定性。（2）竞争物种的随机迁移形成了源一汇结构,对物种竞争共存具有促进作用,但弱的资源利用者（较高的R^＊）的迁移率不宜过高。（3）在种群统计率相同的条件下,资源增长率差异越大,越不利于消费者物种的共存;若种群统计率不相同,在资源增长率相同的情况下,物种共存又是不可能的,在自然界中,物种共存需要资源增长率的差异。（4）不同类型的资源增长对竞争物种的稳定性的影响是不同的。     

Studies on inoculation of aerial algae. I. Semi-mass culture and quantification of algal color of Klebsormidium flaccidum (Ulotrichales, Chlorophyceae) sprayed on to concrete board

This paper describes methods of semi-mass culture and the quantification of algal color sprayed on a concrete board of the alga Klebsormidium flaccidum (Küitzing) S. Mattox et Blackwell. The alga was cultured in 100 L tanks normally used for hatching brine shrimp, with aeration from the deepest parts of the slanting sides. Initially mean algal density increased rapidly and attained 9.1 times the original density after 4 weeks. Subsequently, increase of the algal density was slow, and the density became 16.1 times the original level after 8 weeks. The alga cultured in this study was sprayed on to a concrete board using an agricultural sprayer. The relationship between the algal density on the board and its color, expressed as L*a*b* color space, was quantified. The chromaticity was maximum as the algal density on the board was about 5 g/m², and the color was most vivid. When the algal density was more than 20 g/m², both the lightness and chromaticity changed slightly, and the color became dark green.     

云南地区恒河猴MHC部分基因频率及多态性的分析

目的不同种群恒河猴（Macaca mulatta）主要组织相容性复合体（major histocompatibility complex,MHC）基因的差异性,很大程度上影响了用其作为动物模型的实验研究结果的稳定性、可靠性和可重复性。清晰了解云南地区恒河猴种群免疫遗传学背景对于运用该种群开展各项研究非常重要。恒河猴基因Mamu（Macaca mulatta）-A＊01就被公认与猴免疫缺陷病毒（SIV）的感染与延缓病程相联系。Mamu-Ⅱ类等位基因Mamu-DRB1＊0306、0309有研究表明也与SIV感染后的病程发展有关。方法针对目前研究热点的Mamu-A＊01基因及Ⅱ类Mamu-DRB1＊0306、0309基因,采用序列特异性引物-聚合酶链式反应方法（PCR-SSP）,对云南地区的140只恒河猴（其中92只来源于云南景东野外自然繁殖群、48只来源于本灵长类中心自繁群）进行检测。并对阳性样本用部分测序结合单链构象多态性分析（SSCP）的方法加以验证。结果Mamu-A＊01检测结果为阴性。Mamu-DRB1＊0306、0309基因频率分别为32%及45%。结论初步显示云南地区恒河猴种群Mamu-A＊01基因可能缺失或基因频率很低,但存在Mamu-DRB1＊0306、0309基因。初步证明SSCP方法代替大规模测序是可行的,为云南地区恒河猴在AIDS研究中的应用提供了部分依据,一定程度上填补了该种群的遗传背景资料。